The goals of this research proposal are to develop electrochemistry-based probes and ancillary tools to monitor neurotransmitters and other chemical substances in intact, functional brain tissue. Real-time measurements of chemical changes in the brain enable the regulatory kinetics and mechanisms to be . unravelled, and they also allow investigation of the alteration of these mechanisms by disease or drugs of abuse. Carbon-fiber microelectrodes will be employed with fast-scan cyclic voltammetry. Developed in the previous project periods, this approach provides a dynamic view of neurotransmitters relaying information in the brain. The major target neurotransmitter is dopamine, and the metabolic indicators, molecular oxygen and local pH, will also be monitored. The specific aims involve new technology (Aims 1, 2, and 5) and innovative applications (Aims 3 and 4). They are: 1. To employ principle component analysis of cyclic voltammograms collected in vivo to extract the major contributors to the electrochemical signal. This will improve signal-to-noise ratios and will separate pH and dopamine changes. Its use will allow further insight into the changes induced by cocaine and cannabinoids. 2. To develop and use iontophoresis with voltammetry to introduce chemical substances directly at the site where pH and dopamine measurements are made. This will allow local mechanisms that regulate dopamine release and uptake to be probed. In freely moving animals, the local injections will avoid whole animal effects of injected pharmacological agents. 3. To investigate dopamine release from its cell bodies. Measurements will be made in the ventral tegmental area while antidromically activating dopamine fibers. The mechanisms that control release and uptake will be contrasted with those in the terminal regions. 4. To investigate dopamine neurotransmission and pH changes in a model of a disease state. R6/2 mice, a genetic model of Huntington's disease, will be used. Preliminary data indicate dopamine neurotransmission is impaired in these animals. We will investigate the origin of this impairment and probe regulation of 5-hydroxytryptamine neurotransmission and regulatin of pH. 5. To develop carbon electrodes with greater flexibility and less fragility. The proposed design uses various forms of carbon deposited on the tips of tungstem wires. These electrodes will facilitate measurements in behaving animals and the development of arrays of electrodes for in vivo use.